Human tumor cells possess a number of characteristics that differ from their normal counterparts. Understanding the molecular details of these differences is critical to our understanding of the tumorigenic process and the rational development of novel cancer therapeutics. Acquisition of immortality is one attribute that is required in the development of a human tumor cell. Cellular immortality is the result of stable telomere maintenance in the face of ongoing cellular division. The telomere is a protective DNA-protein structure located at the end of all eukaryotic chromosomes. In normal cells continued rounds of replication result in telomere shortening that eventually leads to cell arrest. In contrast, tumor cells maintain stable telomere lengths through either the activation of the reverse transcriptase, telomerase, or through an alternative telomere maintenance mechanism (ALT). We have demonstrated that in telomerase-positive cell lines, which represent approximately 90% of all cancers, inhibition of telomerase activity results in loss of telomere maintenance and cell death. These studies also demonstrated that such inhibitors have no affect on telomerase- negative ALT cells. Currently the molecular details of the ALT pathway are unknown. Therefore, in this proposal we seek to unravel the molecular details of the ALT mechanism through the use of a non- biased retroviral screen. In addition, we have shown that in some cells ALT can not substitute for telomerase in tumorigensis raising the question of what role telomerase plays in tumorigenesis in addition to telomere length maintenance. One possibility is that in addition to telomere length telomerase may also aid in maintaining the correct telomere structure and that in some cases ALT may by insufficient. Therefore, we will examine the structure of the telomere in ALT cells in the presence or the absence of telomerase. The specific aims of this proposal are: 1. Identify the gene(s) responsible for telomerase-independent telomere maintenance. 2. Examine the role of ALT and telomerase in tumorigenesis. 2. Examine the role fo ALT and telomerase in tumorigenesis.